Method of regulating gene expression

ABSTRACT

The present invention relates, in general, to gene expression and, in particular, to a method of inhibiting the expression of a target gene and to constructs suitable for use in such a method.

This application is a divisional of U.S. application Ser. No.14/568,680, filed Dec. 12, 2014 and is a continuation of U.S.application Ser. No. 13/737,662 (published as US 2013-0191935 A1), filedJan. 9, 2013, both of which claim priority to U.S. application Ser. No.13/356,514, filed Jan. 23, 2012 (now U.S. Pat. No. 8,409,796 issued Apr.2, 2013), which is a continuation of U.S. application Ser. No.10/429,249, filed May 5, 2003, (now U.S. Pat. No. 8,137,910 issued Mar.20, 2012), which claims priority from Provisional Application No.60/377,224, filed May 3, 2002, the entire contents of each of which arehereby incorporated by reference.

TECHNICAL FIELD

The present invention relates, in general, to gene expression and, inparticular, to a method of inhibiting the expression of a target gene,to constructs suitable for use in such a method and to plants andnon-human animals comprising such constructs. The invention also relatesto compositions and kits comprising constructs that can be used toinhibit gene expression.

BACKGROUND

Animal cells have recently been shown to express a novel class ofsingle-stranded, ˜22 nucleotide (nt) non-coding RNAs, termed micro RNAs(miRNAs) (Lagos-Quintana et al, Science 294:853-858 (2001); Lau et al,Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001)).miRNAs appear to be derived from ˜70 nt precursors that form a predictedRNA stem-loop structure. It remains unclear whether these miRNAprecursor molecules are transcribed from autonomous promoters or areinstead contained within longer RNAs (Ambros, Cell 107:823-826 (2001);Lau et al, Science 294:858-862 (2001)).

While over 100 distinct miRNAs are expressed in organisms as diverse asnematodes (Lau et al, Science 294:858-862 (2001), Lee et al, Science294:862-864 (2001)), fruit flies (Lagos-Quintana et al, Science 294858-858 (2002), and humans (Mourelatos et al, Genes Dev. 16:720-728(2002)), as well as in plants (Tang et al, Genes Dev. 17:49-63 (2003),Reinhart et al, Genes Dev. 16:1616-1626 (2002)), their function remainslargely uncertain. However, the biological activity of two miRNAs, C.elegans let-7 and lin-4, is well established (Lee et al, Cell 75:843-854(1993); Reinhart et al, Nature 403:901-906 (2000)). Both lin-4 and let-7are expressed during specific larval stages and both miRNAs interactwith partially complementary RNA targets, located in the 3′ untranslatedregion (3′ UTR) of specific mRNAs, to selectively block theirtranslation. This inhibition is important for appropriate developmentalregulation in C. elegans (Wightman et al, Cell 75:855-862 (1993); Slacket al, Mol. Cell 5:659-669 (2000)).

Several miRNAs, including let-7, are evolutionarily conserved from C.elegans to man, as are several let-7 targets (Ambros, Cell 107:823-826(2001)). This conservation implies that let-7, as well as other miRNAs,may also repress the expression of specific mRNA species in mammaliancells. This hypothesis is also suggested by the similarity betweenmiRNAs and small interfering RNAs (siRNAs), ˜21 nt double-stranded RNAsthat can induce the degradation of mRNA molecules containing perfectlymatched complementary targets, a process termed RNA interference (RNAi)(reviewed by Sharp, Genes Dev. 15:485-490 (2001), see also Hutv<gner etal, Curr. Opin. Genet. Dev. 12:225-232 (2002) and Zamore et al, Science296:1265-1269 (2002), further see U.S. Pat. No. 6,506,559). However,while miRNAs are encoded within the host genome, siRNAs are generallyexcised from larger dsRNA precursors produced during viral infection orintroduced artificially.

Because the introduction of artificial siRNAs into animal cells caninduce the degradation of homologous mRNA molecules, RNAi has emerged asa useful experimental tool (Elbashir et al, Nature 411:494-498 (2001);Fire et al, Nature 391:806-811 (1998); Hammond et al, Nature 404:293-295(2000)). However, in mammalian cells, induction of RNAi required thetransfection of RNA oligonucleotides, which can be inefficient and givesrise to only a transient inhibition in target gene expression.

The present invention provides RNA molecules (miRNAs) functionallyequivalent to siRNAs that can be transcribed endogenously in animal andplant cells. The invention makes possible the production of miRNAsspecifically designed to inhibit the expression of mRNA containing acomplementary target sequence. The miRNA molecules of the invention canbe used experimentally or therapeutically to inhibit gene function.

SUMMARY OF THE INVENTION

The present invention relates to artificial miRNAs and to a method ofusing same to specifically inhibit the expression of selected genes inhuman and non-human animal cells and in plant cells. In accordance withthe invention, an miRNA-encoding DNA sequence is introduced into thecells and inhibition of the target gene is induced by endogenouslytranscribed miRNAs. Where advantageous, transcription of the miRNA canbe placed under the control of an inducible promoter or a tissuespecific promoter. As the present method can result in continuous miRNAproduction, stable inhibition of target mRNA expression can be effected.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Production of the miR-30 miRNA in transfected cells. (FIG.1A) (SEQ ID NO:5) Diagram of the predicted human miR-30 precursor RNA(Lagos-Quintana et al, Science 294:853-858 (2001)). Mature miR-30 (3′arm) and anti-miR-30 (5′ arm) are indicated lines. Arrows point to the5′ ends of the mature miRNA as determined by primer extension analysis.The position of the 3′ ends may have an error of 1 nucleotide. (FIG. 1B)Northern blot analysis of miR-30 and anti-miR-30 in transfected 293Tcells. Lanes 1 and 4: RNA from mock-transfected 293T cells. Lanes 2 and5: cells transfected with pCMV-miR-30. Lanes 3 and 6: cells transfectedwith pCMV-mmiR-30 (mmiR=mature miR). The relative mobility of syntheticDNA oligos is indicated. “*” indicates the position of a suspectedendogenous anti-miR-30 species.

FIGS. 2A-2C. The miR-30 miRNA selectively inhibits expression of anindicator mRNA containing miR-30 target sites. (FIG. 2A) (SEQ ID NOs:6and 7) The sequence of a designed target site partially complementary tomiR-30. pDM128/RRE/4XT was derived from pDM128/RRE by insertion of fourcopies of this target site into the 3′ UTR (black boxes). Splice sites(ss), the RRE and the relative position of the RPA probe are indicated.(FIG. 2B) 293T cells were co-transfected, with 10 ng of an internalcontrol plasmid (pBC12/CMV/β-gal) expressing β-galactosidase (β-gal)and, as indicated, 10 ng of pDM128/RRE or pDM128/RRE/4XT, 10 ng pcRev,and 400 ng of pCMV-mmiR-30 or pCMV-miR-30. The parental pBC12/CMVplasmid served as the negative control. CAT activities were determinedat 48 hrs postransfection and were normalized for β-gal activities.Columns 2 and 6 are arbitrarily set at 100%. (FIG. 2C) 293T cells weretransfected with the pDM128/RRE/4XT plasmid, with or without pcRev orpCMV-miR-30, as described in FIG. 2B. At 48 hr. after transfection,cells were divided into nuclear (N) and cytoplasmic (C) fractions, totalRNA isolated and analyzed by RPA. The probe fragments rescued by thespliced (S) and unspliced (U) mRNAs encoded by pDM128/RRE/4XT areindicated.

FIGS. 3A-3D. The novel miR-30-nxt miRNA specifically inhibits thecytoplasmic expression of unspliced pgTAT-nxt mRNA. (FIG. 3A) (SEQ IDNO:8) Design of the precursor of the miR-30-nxt miRNA. Insertedsequences derived from the global Drosophila nxt gene are indicated.(FIG. 3B) Detection of the novel miR-30-nxt and anti-miR-30-nxt miRNAsin transfected 293T cells by Northern analysis. Lanes 1 and 3:mock-transfected cells; lanes 2 and 4: pCMV-miR-30-nxt transfectedcells. The relative mobility of DNA markers is indicated. (FIG. 3C)Western blots using rabbit polyclonal antisera directed against HIV-1Tat or Rev. 293T cells were transfected using 25 ng of pgTAT orpgTAT-nxt, 25 ng of pcRev, and 400 ng of pCMV-miR-30-nxt. The parentalpBC12/CMV plasmid served as negative control. This Western analysis wasperformed ˜48 hrs. after transfection. (FIG. 3D) The miR-30-nxt miRNAreduces the cytoplasmic level of unspliced pgTAT-nxt mRNA. 293T cellswere transfected with pgTAT-nxt, with or without pcRev orpCMV-miR-30-nxt. Two days after transfection, nuclear (N) andcytoplasmic (C) RNAs were prepared and analyzed by RPA. Lane 1represents approximately 3% of input (I) probe. Probe fragments rescuedby spliced (S) and unspliced (U) mRNA are indicated.

FIGS. 4A-4D. Inhibition of endogenous gene expression by novel miRNAs in293T cells. (FIG. 4A) Detection of miR-30-PTB and anti-miR-30-PTBexpression. 293T cells were mock transfected (lanes 1 and 3) ortransfected with pCMV-miR-30-PTB (lanes 2 and 4). After 2 days, totalRNA was isolated and used for primer extension analysis. Positions ofDNA markers are indicated. (FIG. 4B) Reduction of endogenous PTB proteinand mRNA expression by pCMV-miR-30-PTB. Cells were transfected withpCMV-miR-30-nxt (lanes 1 and 3) or pCMV-miR-30-PTB (lanes 2 and 4).After five days, total cell lysates and RNAs were prepared. Lanes 1 and2: Western blot using antibodies directed against PTB or CA 150, whichserved as a loading control. Lanes 3 and 4: Northern analysis for PTBmRNA. (FIG. 4C) Loss of SV40 Tag in cells transfected withpCMV-miR-30-Tag. Cells were co-transfected with phrGFP-C (a greenfluorescent protein expression plasmid) and pCMV-miR-30-nxt orpCMV-miR-30-Tag, and three days later, analyzed by immunofluorescence.(FIG. 4D) Quantitation of cells expressing SV40 Tag. Cells with clearnuclear Tag staining were counted as positive (cytoplasmic staining wasweak and also present in secondary antibody-only controls). At least 200cells were counted for each sample.

FIGS. 5A (SEQ ID NOs:9-25, respectively) and 5B. Indicator constructdesign. (FIG. 5A) Sequences of the synthetic RNA targets used and theirpredicted pairing with the miR-30, anti-miR-30 or miR21 miRINA or thedNxt siRNA. Target sequences were either perfectly (P) complementary orwere designed to form a central 3 nt bulge (B). A random sequence, forwhich no complementary small RNA is known to exist, was used as acontrol. (FIG. 5B) Structure of the pCMV-luc-Target andpCMV-luc-Target-CAT indicator constructs. The Targets, represented byblack boxes, are eight tandem repeats of one of the sequences shown inFIG. 5A. PA, polyadenylation signal.

FIGS. 6A-6C. Biological activity of the miR-30 and anti-miR-30 miRNAs.(FIG. 6A) The level of expression of miR-30, anti-miR-30 and of miR-21in mock transfected 293T cells, or in 293T cells transfected with theindicated miRNA expression plasmids, was determined by primer extension(Zeng et al, RNA 9:112-123 (2003)). (FIG. 6B) The luc enzyme activitiesdetected in 293T cell cultures transfected with the listed indicator andeffector plasmids, as well as the pBC12/CMV/β-gal control plasmid, weredetermined ˜40 hr after transfection and then adjusted based on minorvariations observed in the CAT internal control. These values arepresented normalized to the culture transfected with pCMV-luc-random-CATand pCMV-miR-21, which was arbitrarily set at 1.0. Average of threeindependent experiments with standard deviation indicated. The number ofnanograms of each miRNA expression plasmid transfected into each cultureis indicated. (FIG. 6C) Parallel northern analysis to detect the lucreporter miRNA (top panel) and the control β-gal mRNA (bottom panel).Shown above the top panel are the amounts of pCMV-miR-30 or pCMV-miR-21transfected per culture. The level of luc enzyme activity detected foreach indicator construct is given as a percentage of the level obtainedupon co-transfection with the pCMV-miR-21 control plasmid. Lane 1: RNAfrom mock transfected 293T cells. The arrow indicates the position ofthe 1.8 kb luc mRNA cleavage product.

FIGS. 7A and 7B. Biological activity of the human miR-21 miRNA. (FIG.7A) This experiment was performed as described in FIG. 6B. Data shownare the average of 4 independent experiments. (FIG. 7B) Parallelnorthern analysis of luc (upper panel) and β-gal (lower panel) mRNAexpression. The level of luc enzyme activity detected with eachindicator construct is given as a percentage of the level obtained uponco-transfection with the pCMV-miR-30 control plasmid. Lane 1, RNA frommock transfected 293T cells. The arrow indicates the position of the˜1.8 kb luc mRNA cleavage product.

FIGS. 8A and 8B. Inhibition of mRNA utilization by a synthetic siRNA.(FIG. 8A) Cultures were cotransfected with one of the three listedindicator plasmids together with the dNxt or dTap siRNA and the pRL-CMVand pBC12/CMV/β-gal internal control plasmids. The amount of each siRNAused is given in picomoles. Approximately 40 hr after transfection,cultures were used for the dual luciferase assay or for RNA isolation.Firefly luc activities were adjusted for minor variations in the Renillaluc internal control and are presented normalized to the activityobserved in the culture transfected with the pCMV-luc-random controlplasmid and the dTap control siRNA, which was set at 1.0. These datarepresent the average of three independent experiments, with standarddeviation indicated. (FIG. 8B) Northern analysis of firefly luc (upperpanel) and β-gal (lower panel) mRNA expression. The level of firefly lucenzyme activity detected for each indicator construct is given as apercentage of the level obtained with the dTap control siRNA. Lane 1,RNA from a mock transfected culture. The arrow indicates the position ofthe ˜1.8 kb luc mRNA cleavage product.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of specifically inhibiting theexpression of selected target genes in human and non-human animal cellsand in plant cells using endogenously produced miRNA. In accordance withthis method, constructs are used that encode one or multiple miRNAs. Theconstructs are designed such that nuclear processing, transport andexcision of mature miRNA are effected efficiently. The resulting miRNAinduces degradation of an mRNA produced in the cell that contains acomplementary target sequence or otherwise inhibits translation of themRNA. The invention further relates to constructs suitable for use insuch a method and to compositions and kits comprising such constructs.

In accordance with the present method, a DNA construct is introducedinto cells (host cells) in which a target gene sequence is expressed.The construct comprises a promoter functional in the host cells operablylinked to a sequence encoding a precursor of the miRNA. Introduction ofthe construct into the host cells is effected under conditions such thatthe miRNA precursor transcript is produced and mature miRNA is thenexcised from the precursor by an endogenous ribonuclease. The resultingmature miRNA induces degradation of the mRNA transcript of the targetgene sequence produced in the cell or otherwise inhibits translation ofthe mRNA. (It will be appreciated that degradation of other types ofRNA, including viral RNA, can be similarly induced.)

miRNAs suitable for use in the present invention are, advantageously,about 19-24 nucleotides long, preferably, about 21 or 22 nucleotides inlength. The miRNAs can be designed so as to hybridize to any RNAtranscript with a high degree of specificity. Advantageously, the miRNAis designed so as to be perfectly complementary to the target sequencewithin the RNA (e.g., mRNA) as even a single nucleotide reduction incomplementarity to the target can, depending on its location, attenuatethe level of inhibition. The data presented in Example 2 indicate thatmiRNA can cleave mRNA bearing a fully complementary target site whilemiRNA can inhibit expression of mRNA bearing partially complementarysequence without necessarily inducing cleavage. The miRNA can bedesigned so as to target a 3′ or 5′ untranslated region of the mRNA orcoding region of the mRNA.

As indicated above, the miRNA is excised from a precursor that includesa predicted RNA stem-loop structure (Lagos-Quintana et al, Science294:853 (2001), Lau et al, Science 294:858 (2001), Lee and Ambrose,Science 294:362 (2001)). This structure stem-loop can be designed suchthat it is recognized by a ribonuclease (e.g., an RNAse III-type enzyme,such as DICER, or an enzyme having the recognition properties thereof),with the resulting excision of the mature miRNA. Such precursorstem-loop structures can be about 40 to 100 nucleotides long,preferably, about 50 to 75 nucleotides. The stem region can be about19-45 nucleotides in length (or more), preferably, about 20-30nucleotides. The stem can comprise a perfectly complementary duplex (butfor any 3′ tail), however, “bulges” can be present on either arm of thestem and may be preferred. Advantageously, any such “bulges” are few innumber (e.g., 1, 2 or 3) and are about 3 nucleotides or less in size.The terminal loop portion can comprise about 4 or more nucleotides(preferably, not more than 25); the loop is preferably 6-15 nucleotidesin size. The precursor stem loop structure can be produced as part of alarger, carrier transcript from which the miRNA is excised, or it can beproduced as a precise transcript.

The data presented in Zeng et al, RNA 9:112-123 (2003), make clearcertain sequence requirements for efficient miRNA processing andfunctioning (for example, maintenance of base-pairing at the base of thepredicted stem, outside the stem portion encoding mature miRNA, beingsignificant), those requirements being incorporated herein by reference.The data presented also demonstrate the desirability of substitutingstem sequences of naturally occurring miRNAs (e.g., miR-30) to generatemiRNAs suitable for use in inhibiting expression of any target gene. Thedata indicate that while the presence of a miR-30 loop may be desirable,variations of that structure can also be tolerated (e.g., loops can beused that are greater than 72%, preferably greater than 79%, morepreferably greater than 86%, and most preferably, greater than 93%identical to, for instance, the miR-30 sequence (determinedconventionally using known computer programs such as the BESTFIT program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711)).

The encoding sequence of the invention (e.g., the miRNA precursorencoding sequence or longer carrier encoding sequence) can be present inthe construct in operable linkage with a promoter. Appropriate promoterscan be selected based on the host cell and effect sought. Suitablepromoters include constitutive and inducible promoters, such asinducible RNA polymerase II (polII)-based promoters. The promoters canbe tissue specific, such promoters being well known in the art. Examplesof suitable promoters include the tetracycline inducible or repressiblepromoter, RNA polymerase I or III-based promoters, the pol II dependentviral promoters such as the CMV-IE promoter, and the polIII U6 and HIpromoters. The bacteriophage T7 promoter can also be used (in whichcase, it will be appreciated, the T7 polymerase must also be present).

The constructs of the invention can be introduced into host cells usingany of a variety of approaches. Infection with a viral vector comprisingthe construct can be effected. Examples of suitable viral vectorsinclude replication defective retroviral vectors, adenoviral vectors,adeno-associated vectors and lentiviral vectors. Transfection with aplasmid comprising the construct is an alternative mode of introduction.The plasmid can be present as naked DNA or can be present in associationwith, for example, a liposome. The nature of the delivery vehicle canvary with the host cell.

In vivo delivery of the construct (e.g., present in a viral vector) canbe carried out using any one of a variety of techniques, depending onthe target tissue. Delivery can be, as appropriate, by direct injection,inhalation, intravenous injection or other physical method (includingvia microprojectiles to target visible and accessible regions of tissue(e.g., with naked DNA)). Administration can be by syringe needle,trocar, canula, catheter, etc., as appropriate.

The miRNAs of the invention can be used to regulate (e.g., inhibit)expression of target genes in cells and tissues in culture and in cellspresent in plants and in humans and non-human animals. The targetsequences can be naturally occurring sequences, transgenes or can bepathogen sequences present, for example, as a result of infection. Asone example, miRNAs of the invention can be used to “turn off” papillomaviruses in humans (e.g., in the uterus by using an appropriatelydesigned adeno-associated viral vector).

Cultured cells suitable as hosts in accordance with the inventioninclude both primary cells and cell lines. The cells can be human cells,including human stem cells. A construct of the invention encoding anmiRNA can be introduced into cultured cells to inactivate a specificgene of unknown function. Silencing the gene using the method of theinvention can be used as an approach to assess its function.Alternatively, a construct encoding an miRNA can be introduced intocells to be implanted into a human or non-human animal for therapeuticpurposes. For example, hepatic stem cells can be obtained from a patientinfected with hepatitis C and placed in culture. A construct of theinvention encoding an miRNA that targets a gene of hepatitis C essentialto, for example, replication or packaging can be introduced into theexplanted cells under conditions so that the gene is silenced. The cellscan then be reimplanted into the patient under conditions such thatregeneration is effected.

miRNAs of the invention can also be introduced into a non-human animalto produce a model experimental animal, or into a human or non-humananimal for therapeutic purposes. In the case of experimental animals,the miRNAs can be used for large scale analysis of gene function. As thetarget for the miRNA is about 22 nucleotides, the miRNAs can be used toknockout expression of individual isoforms resulting, for example, fromalternative splicing. In the case of therapy, miRNAs can be designed,for example, to block viral replication. Human and non-human animals canbe engineered, for example, to permanently express multiple miRNAstargeted to conserved sequences in viruses (e.g., packaging sequences orregulatory elements), thus rendering the humans/animals permanentlyimmune to virus challenge, including HIV challenge. Similar approachescan be used in plants to render plants immune to viruses.

Appropriately designed miRNAs can also be used in humans and non-humananimals to turn off oncogene expression in tumor cells, or inhibitexpression of genes associated with other medical conditions, e.g.,mutant forms of Huntingtin or of the prion protein as well as dominantnegative protein mutants seen in some human genetic diseases. miRNAs ofthe invention can be used, for example, to inhibit expression ofpro-inflammatory genes or apoptosis genes where therapeuticallydesirable. For instance, expression of BCL-2 can render tumor cellsresistant to chemotherapy. Using the present approach, miRNAs can beused to inhibit expression of BCL-2 and enhance the ability ofchemotherapeutic agents to cause tumor cells to undergo senescence.Similarly, T cells isolated from a tumor bearing patient can be modifiedex vive using the present approach to such that expression of the TGF∃receptor is inhibited. Upon reintroduction into the patient, the killingability of the T cells is enhanced. Likewise, T cells can be modified exvivo to inhibit expression of the Fas receptor, thereby increasing thetumor killing capacity of the cells upon reintroduction. MiRNAs of theinvention can be used to treat any disease where turning down one or aset of specific gene products is beneficial.

The miRNAs of the invention can also be used to carry out various highthroughput screens to select for loss of function phenotype. Forexample, a library of random miRNA precursor-encoding constructs can beintroduced into cells (e.g., using a viral vector) to determine functionof a genomic sequence. Typically, the protocol used is such that virusis introduced per cell. Using any of a variety of approaches, thosecells in which the function of the targeted gene is lost can be selected(e.g., if a gene involved in cell death resulting from viral infectionis sought, only those cells that contain the targeting miRNA will remainviable after exposure to the virus; alternatively, markers (e.g.,indicator proteins) can be used to select for cells containing thetargeting miRNA). The miRNA can then be cloned out of the selectedcells, the sequence determined and used for identifying the targetedgene.

The present invention includes compositions and kits comprising theabove-described miRNAs and/or nucleic acid sequences encoding same (andconstructs comprising such nucleic acids). Such compositions can furtherinclude, for example, a carrier (e.g., a sterile carrier) and such kitscan further comprise, for example, ancillary reagents (e.g., buffers)such as those necessary to carry out the instant methods, and containermeans′.

Certain aspects of the invention are described in greater detail in thenon-limiting Examples that follow (see also Zeng et al, Mol. Cell9:1327-1333 (2002), Coburn et al, J. Virol. 76:9225-9231 (2002) and Zenget al, RNA 9:112-123 (2003), as well as U.S. Pat. No. 6,506,559, e.g.,for specific applications).

EXAMPLE 1 Experimental Procedures

Plasmid Construction and Oligonucleotide Description

The expression plasmids pBC12/CMV, pBC12/CMV/β-gal and pcRev, and theindicator constructs pDM128/RRE and pgTat, have been previouslydescribed (Malim et al, Nature 338:254-257 (1989); Bogerd et al, Crml.J. Virol. 72:8627-8635 (1998); Hope et al, Proc. Natl. Acad. Sci. USA87:7787-7791 (1990); Cullen, Cell 46:973-982 (1986)). A GPP expressionplasmid, phrGFP-6, was obtained from Strategene. To make pCMV-miR-30,the two DNA primers:

(SEQ ID NO: 1 ) 5′-TACTCGAGATCTGCGACTGTAAACATCCTCGACTGGAAGCTGTGAAGCCACAGATGG-3′ and (SEQ ID NO: 2)5′-CGCTCGAGGATCCGCAGCTGCAAACATCCGACTGAAAGCCCATCTG TGGCTTCACAG-3′were annealed, extended using Taq DNA polymerase, cut with XhoI, andcloned into the XhoI site present in the pBC12/CMV. To make pCMV-miR-30,5′-ATCCCTTTCAGTCGGATGTTGCAGCT-3′ (SEQ ID NO:3) and5′-CTAGAGCTGCAAACATCCGACTGAAAGG-3′ (SEQ ID NO:4) were annealed andcloned into pBC12/CMV. To make pDM128/RRE/4XT, four copies of the miR-30target site (FIG. 2A, separated by two or five nucleotides from eachother) were cloned into the XhoI site of pDM128/RRE. To make pgTAT-nxt,the Drosophila nxt-coding sequence (nucleotides 1-420) were amplifiedfrom a Drosophila embryonic cDNA library and cloned between the twoBglII sites present in pgTAT. The pCMV-miR-30-PTB, pCMV-miR-30-nxt andpCMV-miR-30-TAg expression plasmids were prepared as described forpCMV-miR-30, except that the inserted stem sequences were derived fromeach target gene.Cell Culture and Transfection

293T cells were grown as previously described (Bogerd et al, Crml. J.Virol. 72:8627-8635 (1998)) and were transfected using FuGene 6 Reagent(Roche). CAT assays were performed at 48 hrs. after transfection, asdescribed (Bogerd et al, Crml. J. Virol. 72:8627-8635 (1998)). ForWestern blotting, lysates were fractionated on a 4-20% SDS-acrylamidegradient gel (Bio-Rad), transferred, and then probed with a rabbitpolyclonal antiserum directed against Tat, Rev (Malim et al, Nature338:254-257 (1989)), CA 150 (Suné et al, Mol. Cell. Biol. 17:6029-6039(1997)) or PTB. Reactive bands were visualized using ECL (Amersham). Apolyclonal antiserum specific for human PTB 1 was prepared byimmunization of rabbits with a purified recombinant fusion proteinconsisting of glutathione-S-transferase fused to full length PTB1.Immunofluorescence analyses were performed as described (Wiegand et al,Mol. Cell. Biol. 22:245-256 (2002)) using a monoclonal antibody againstSV40 Tag (Pab 108, Santa Cruz) and rhodamine-conjugated goat anti-mouseantiserum (ICN) as well as the DNA strain DAPI.

RNA Analysis

Total RNA was isolated using Trizol Reagent (Invitrogen). Cellfractionation and RPA were performed as previously described (Kang andCullen, Genes Dev. 13:1126-1139 (1999)). For miRNA Northern analysis,approximately 20 j g of total RNA was separated on a denaturing 15%polyacrylamide gel, transferred to a HyBond-N membrane (Amersham), UVcrosslinked, and probed with 5′ ³²P-phosphorylated oligos in ExpressHybsolution (Clontech). For Northern analysis of mRNA, 20 g of total RNAwas fractionated on a 1% denaturing agarose gel, transferred tomembrane, fixed, and probed with a random primed PTB cDNA probe.

Results

Expression of an Introduced miR-30 miRNA Sequence in Human Cells

MiR-30 is one of several novel miRNAs recently isolated from the humancell line HeLa (Lagos-Quintana et al, Science 294:853-858 (2001)). AcDNA sequence encoding the entire predicted 71 nt miR-30 precursor (FIG.1A) was cloned into the context of an irrelevant mRNA expressed underthe control of the cytomegalovirus immediate early (CMV-IE) promoter, inpCMV-miR-30. A similar plasmid, pCMV-miR-30, containing only the maturemiR-30 cDNA sequence was also constructed. Human 293T cells were thentransfected with these expression plasmids and total RNA was analyzedfor the presence of the miR-30 miRNA by Northern blotting (FIG. 1B).Mature miR-30 could be readily detected in cells transfected withpCMV-miR-30 (FIG. 1B). The miRNA produced from the transfectedpCMV-miR-30 plasmid appeared to be ˜22 nt in length and had the same 5′end as reported for endogenous miR-30 (Lagos-Quintana et al, Science294:853-858 (2001)), as determined by primer extension analysis (FIG.1A). In contrast, mock-transfected or pCMV-miR-30 transfected 293T cellsexpressed no detectable miR-30 miRNA (FIG. 1B, lanes 1 and 3).Production of the miR-30 miRNA could also be detected in transfectedHeLa or NIH3T3 cells or when the miR-30 precursor DNA was placed withinan intron or in the 3′-UTR of another mRNA expressed under the controlof the CMV-IE promoter. Thus, the mature miR-30 miRNA can be excisedfrom the miR-30 precursor sequence when the latter is expressed withinthe context of an irrelevant mRNA.

Mature miR-30 is encoded by the 3′ arm of its precursor (FIG. 1A), andone miRNA precursor generally gives rise to only one stable, maturemiRNA species, derived from either the 5′ or 3′ arm of the precursor RNAhairpin (Lagos-Quintana et al, Science 294:853-858 (2001); Lau et al,Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001)).Nevertheless, it was possible to also detect a miRNA derived from the 5′arm (antisense miR-30, or anti-miR-30) in transfected cells (FIG. 1B.lane 5). While significant levels of endogenous miR-30 miRNA were notdetected in either 293T cells or, surprisingly, HeLa cells, thereappeared to be a low, constitutive level of endogenous anti-miR-30, orpossibly of a similar miRNA, in 293T, HeLa and NIH3T3 cells (marked by“*” in FIG. 1B).

MiR-30 Inhibits the Expression of an mRNA Containing ComplementaryTarget Sites

The C. elegans miRNAs lin-4 and let-7 inhibit the translation of mRNAscontaining multiple complementary sequences in their 3′ UTRs withoutsignificantly affecting the steady-state level of the miRNA (Lee et al,Cell 75:843-854 (1993); Wightman et al, Cell 75:855-862 (1993)). It wastherefore questioned whether human miR-30 could also act via a similarmechanism. A miR-30 target sequence was designed, and four copies ofthis sequence were inserted into the 3′ UTR of the indicator constructpDM128/RRE to give the pDM128/RRE/4XT plasmid (FIG. 2A). Importantly,this target sequence is not a perfect complement to miR-30 and instead,like known lin-4 and let-7 targets (Lee et al, Cell 75:843-854 (1993);Slack et al Mol. Cell 5:659-669 (2000)), contains a central mismatch(FIG. 2A).

The parental pDM128/RRE indicator construct used in these experimentscontains 5′ and 3′ splice sites flanking an intron, derived from humanimmunodeficiency virus type 1 (HIV-1), that contains both the cat geneand the Rev Response Element (RRE) (Hope et al, Proc. Natl. Acad. Sci.USA 87:7787-7791 (1990)). As previously shown (Hope et al, Proc. Natl.Acad. Sci. USA 87:7787-7791 (1990); Bogerd et al, Crml. J. Virol.72:8627-8635 (1998); Kang and Cullen, Genes Dev. 13:1126-1139 (1999));nuclear export and of this unspliced cat mRNA is dependent onco-expression of the HIV-1 Rev protein, while nuclear export of thespliced mRNA encoded by pDM128/RRE, which does not encode CAT, occursconstitutively (FIG. 2). As shown in FIG. 2B, co-transfection ofpCMV-miR-30, encoding the entire miR-30 RNA precursor, resulted in amarked drop in the level of CAT activity expressed from thepDM128/RRE/4XT plasmid, which contains four copies of the target site,but failed to affect CAT expression from the parental pDM128/RREindicator plasmid (FIG. 2B). In contrast, co-transfection ofpCMV-miR-30, containing only the mature miR-30 sequence, did not reduceCAT expression (FIG. 2B).

To determine whether the observed reduction in CAT activity was due to areduction in cat mRNA expression, an RNase protection assay (RPA) wasperformed using nuclear and cytoplasmic RNA fractions derived from thetransfected 293T cells. As shown in FIG. 2C, miR-30 did notsignificantly affect the cytoplasmic steady-state level of the unsplicedcat mRNA encoded by pDM128/RRE/4XT (compare lanes 6 and 8). Thus, theaction of the miR-30 miRNA in this reporter system appears to mimic theeffect of the lin-4 miRNAs in C. elegans (Olsen and Ambros, Dev. Biol.216:671-680 (1999)).

Designed miRNAs can be Produced In Vivo from Artificial miRNA Precursors

To determine whether the features found in the miR-30 precursor could beused to design and synthesize novel miRNAs in human cells, the stemsequence in the miR-30 precursor was substituted with a sequence basedon the Drosophila nxt gene (Gene CG10174, nucleotides 121-143 from thetranslation initiation codon) (FIG. 3A). It has been previously shownthat analogous synthetic siRNAs can block nxt mRNA expression inDrosophila S2 cells (Wiegand et al, Mol. Cell. Biol. 22:245-256 (2002)).Importantly, this sequence is not conserved in human nxt homologs.

The new miRNA precursor, termed miR-30-nxt, was again expressed as partof a longer mRNA transcript, as described above for wild-type miR-30.Initially, the pCMV-miR-30-nxt plasmid was transfected into human 293Tcells, total RNA isolated, and the production of both the maturemiR-30-nxt miRNA (the 3′ arm, in accordance with miR-30) andanti-mir-30-nxt (the predicted 5′ arm) analyzed by Northern analysis. InFIG. 3B (lanes 2 and 4), it shown that both miR-30-nxt andanti-miR-30-nxt were indeed expressed. Using primer extension analysis,it was possible to determine that the 5′ cleavage sites used in thesynthesis of these novel miRNAs were close to those observed in themir-30 precursor. Thus, novel miRNAs can be produced in human cellsusing the existing, natural miR-30 miRNA precursor as a template.

Inhibition of mRNA Expression by Designed miRNAs

To determine if endogenously transcribed miRNAs could be used as siRNAsto initiate RNAi against specific mRNA targets in mammalian cells, anindicator construct, termed pgTat-nxt, was constructed that contained aninserted 402 nucleotide sequence, derived from the Drosophila nxt gene,that should provide a single, fully complementary target site for thenovel, miR-30-nxt miRNA. The previously described, pgTat indicatorconstruct (Malim et al, Nature 338:254-257 (1989)) contains the twoexons encoding the HLV-1 Tat protein flanking an intron, derived fromthe HIV-1 env gene, that also contains the HIV-1 RRE. In the absence ofRev, pgTat produces exclusively the 86 amino acid (aa), two exon form ofTat encoded by the spliced tat mRNA (FIG. 3C, lane 2). However, in thepresence of the Rev nuclear RNA export factor, the unspliced mRNAencoded by pgTat is also exported from the nucleus, resulting inexpression of the short, 72 as form of the Tat protein (FIG. 3C, lane 3)(Malim et al, Nature 338:254-257 (1989)). Insertion of the nxt sequenceinto the intron of pgTat did not perturb this expression pattern (FIG.3C, lanes 5 and 6). Because the target for pCMV-miR-30-nxt is onlypresent in the intron, expression of miR-30-nxt should only affect theproduction of 72 aa Tat (in the presence of Rev), but not 86 as Tat,thus providing an ideal of control for specificity. This selectiveinhibition was indeed observed (FIG. 3C, compare lanes 6 and 7).Importantly, miR-30-nxt did not inhibit the synthesis of the Revprotein, of the long form of Tat produced by both pgTAT and pgTAT-nxt orof the short, 72 as form of Tat expressed from the pgTAT negativecontrol plasmid (FIG. 3C, lanes 4 and 7).

RNAi induces the degradation of target mRNAs (Hammond et al, Nature404:293-295 (2000); Zamore et al, Cell 101:25-33 (2000)). An RPA wastherefore performed to compare the levels of spliced and unspliced TatmRNAs in the absence or presence of Rev and miR-30-nxt. MiR-30-nxtinduced a specific decrease (˜7 fold) in the cytoplasmic unspliced tatmRNA level seen in the presence of Rev (compare lanes 7 and 9 in FIG.3D), yet it had no effect on spliced tat mRNA. Similar results wereobtained using a synthetic siRNA, thus strongly suggesting that themiR-30-nxt miRNA induces RNAi.

Inhibition of Endogenous Gene Expression Using Artificial miRNAs

To test whether a novel miRNAs could inhibit the expression ofendogenous genes in human cells, the polypyrimidine tract-bindingprotein (PTB) (Wagner and Garcia-Blanco, Mol. Cell. Biol. 21:3281-3288(2001)) was chosen as a target. The pCMV-miR-30-PTB expression plasmid(containing PTB nucleotides 1179-1201), was constructed in the same wayas described for pCMV-miR-30-nxt and transfected into 293T cells. Boththe miR-30-PTB and the anti-miR-30-PTB miRNA were readily detected byprimer extension (FIG. 4A). Importantly, introduction of pCMV-mirR30-PTBresulted in a marked and specific reduction in the level of expressionof the endogenous PTB protein and PTB mRNA, when compared to controlcells (FIG. 4B).

Although introduction of pCMV-miR-30-PTB resulted in a reproducible70-80% drop in the level of PTB protein and mRNA expression intransfected 293T cells (FIG. 4B), inhibition was not complete. Onepossible explanation for the residual level of PTB expression is thattransfection of 293T cells is not 100% efficient. To address thisquestion, a third miRNA expression plasmid, pCMV-miR-30-Tag, wasconstructed that was designed to express an artificial miRNA targetedagainst the SV40 T antigen (Tag) (nt 639-661, Harborth et al, J. CellSci. 114:4557-4565 (2001)). This expression plasmid was then introducedinto 293T cells, which express Tag constitutively, together with aplasmid expressing green fluorescent protein (GFP) and the number of Tagexpressing cells quantitated using immunofluorescence. Co-transfectionof the GFP expression plasmid made it possible to readily discriminatetransfected from non-transfected cells (FIG. 4C). As shown in FIG. 4D,˜90% of cells that were not transfected, or that were transfected withGFP plus pCMV-miR-30-nxt (as a negative control) expressed readilydetectable levels of TAg. In contrast, co-transfection of thepCMV-miR-30-TAg expression plasmid resulted in a dramatic reduction inthe number of cells that were both GFP and TAg positive (FIGS. 4C and4D).

It was subsequently demonstrated that a second human miRNA, termedmiR-21, could also be effectively expressed when the precursor thereforformed part of a longer mRNA (Zeng et al. RNA 9:112-123 (2003)). Forboth miR-30 and miR-21, mature miRNA production was highly dependent onthe integrity of the precursor RNA stem, although the underlyingsequence had little effect.

EXAMPLE 2 Experimental Procedures

Plasmids and siRNAs.

Plasmids pCMV-miR-30, pCMV-miR-21 and pBC12/CMV/β-gal have beendescribed (Zeng et al, RNA 9:112-123 (2003)). Indicator plasmidspCMV-luc-Target (Target being miR30(B), miR-30(AB), miR-30(P),miR-30(AP), miR-21(B), miR-21(P), dNxt(B), dNxt(P) or random, FIG. 5A)were made by combining oligos encoding two copies of the Target sequenceand inserting them after the luciferase (luc) stop codon in pCMV-luc(Zeng et al, RNA 9:112-123 (2003)). At least a 2 bp separation wasintroduced between adjacent target sequences. All plasmids weresequenced to verify the inserted targets. A PCR-amplifiedchloramphenicol acetyl transferase (CAT) expression cassette (FIG. 5B)was then cloned into the unique Stul site present in eachpCMV-luc-Target intermediate. The synthetic dNxt and dTap siRNAs wereobtained from Dharmacon, annealed and stored as 20:M stocks.

Transfections and Reporter Assays.

Transfections were performed in triplicate in 24-well plates. FuGene 6(Roche) was used to transfect plasmids into 293T cells. Each wellreceived 10 ng of pCMV-luc-Target-CAT, 8 ng of pBC12/CMV/β-gal and 400ng of pCMV-miR-30 and/or pCMV-miR-21. For transfections involving bothplasmids and siRNAs, Cellfectamine 2000 (Invitrogen) was used. Each wellreceived 15 ng of pCMVluc-Target, 8 ng of pBC12/CMV/β-gal, 0.2 ng ofpRL-CMV (Promega) and 40 pmol of the dNxt and/or dTap siRNA. 36-44 hourslater, one well of cells was lysed and assayed for firefly luciferaseand either CAT or Renilla luciferase (Zeng et al, RNA 9:112-123 (2003)).RNAs were isolated from the remaining two wells using Trizol Reagent(Invitrogen) or RNAeasy kits (Qiagen). Northern blotting was performedfor at least two independent transfections, as previously described(Zeng et al, RNA 9:112-123 (2003)). The membranes were first hybridizedwith a luc probe, stripped, and then probed for β-galactosidase (β-gal)mRNA.

Results

Previously, it was demonstrated that an indicator gene can betranslationally repressed in human cells upon overexpression of thehuman miR-30 miRNA, if the cognate mRNA bears four tandem copies of abulged RNA target sequence in the 3′UTR (Zeng et al, Mol. Cell9:1327-1333 (2002)). The similar indicator constructs used here arebased on the firefly luciferase indicator gene and contain eight RNAtarget sites tandemly arrayed in the 3′UTR (FIG. 5B). This number iscomparable to the seven target sites for the lin-4 miRNA found in thelin-14 mRNA 3′UTR in C. elegans (Lee et al, Cell 75:843-854 (1993),Wightman et al, Cell 75:855-862 (1993)) and was chosen in the hope ofmaximizing the phenotype of low levels of endogenously expressed miRNAs.The introduced target sites were either perfectly (P) homologous to themiRNAs or siRNAs used, or contained a predicted 3 nucleotide centralbulge (B) (FIG. 5A). An internal control is critical for the experimentsdescribed and initial experiments therefore involved co-transfection ofindicator constructs equivalent to pCMV-luc-Target (FIG. 5B) with acontrol plasmid encoding Renilla luciferase. In light of recent datasuggesting that miRNAs can modulate the chromatin composition of genesbearing homologous DNA sequences (Dernburg et al, Cell 111:159-162(2002)), also constructed was a second set of analogous indicatorconstructs, termed pCMV-luc-Target-CAT, in which the cat gene wasexpressed from a cassette present on the same plasmid (FIG. 5B). Closelysimilar data were obtained using either set of indicator plasmids.

Overexpressed Human miRNAs can Induce mRNA Cleavage.

Although most miRNAs are expressed as single-stranded RNAs derived fromone arm of the pre-miRNA stem-loop structure, a small number ofpre-miRNAs give rise to detectable levels of a miRNA derived from botharms (Lau et al, Science 294:858-862 (2001), Mourelatos et al Genes Dev.16:720-728 (2002)). One such miRNA is human miR-30, and its antisenseform anti-miR-30, both of which have been detected in human cells(Lagos-Quintana et al, Science 294:853-858 (2001), Mourelatos et alGenes Dev. 16:720-728 (2002)). Previously, it was reported that human293T cells do not express detectable miR-30, but do express low levelsof anti-miR-30 (FIG. 6A, lanes 1 and 3) (Zeng et al, Mol. Cell9:1327-1333 (2002)). Transfection of 293T cells with pCMV-miR-30, whichencodes the miR-30 pre-miRNA stem-loop structure contained within alonger transcript (Zeng et al, Mol. Cell 9:1327-1333 (2002)), results inoverexpression of anti-miR-30 and in the production of readilydetectable levels of miR-30 (FIG. 6A, lanes 2 and 4).

To assess the biological activity of these miRNAs, 293T cells weretransfected with indicator constructs analogous to pCMV-luc-Target-CAT(FIG. 5B) containing eight copies of a target sequence perfectlyhomologous to either miR-30 [miR-30(P)] or anti-miR-30 [miR-30(AP)] orsimilar targets predicted to form a central 3 nucleotide RNA bulge[miR-30(B) and miR-30(AB)]. A random 22 nt sequence served as aspecificity control (FIG. 5A). Each indicator construct wasco-transfected with previously described (Zeng et al, RNA 9:112-123(2003), Zeng et al, Mol. Cell 9:1327-1333 (2002)) expression plasmidsencoding either miR-30 (and anti-miR-30) or human miR21, which hereserves as a negative control. In addition, these cells were alsoco-transfected with a plasmid encoding (β-gal.

As shown in FIG. 6B, co-transfection of pCMV-miR-30 suppressed lucexpression from all four indicator constructs bearing either sense orantisense miR-30 RNA targets, when compared to the pCMV-miR-2 1 controlplasmid, but did not affect the control indicator construct bearing therandom target. The two indicator plasmids encoding fully homologous,perfect (P) RNA targets were inhibited significantly more effectivelythan the two constructs encoding partially mismatched, bulged (B) RNAtarget sites when a similar level of the pCMV-miR-30 effector plasmidwas co-transfected. However, equivalent levels of inhibition of lucexpression were achievable by, for example, co-transfecting an ˜10 foldlower level of pCMV-miR-30 with the pCMV-luc-miR-30(P)-CAT indicatorconstruct (FIG. 6B).

The control indicator construct, bearing eight tandem copies of a randomtarget sequence, consistently gave rise to an ˜1.8 fold lower level ofluciferase activity than was seen with the indicator construct bearingthe miR-30 (B) target site in the absence of overexpressed miR-30 miRNA.While not wishing to be bound by theory, it is hypothesized that thislower activity may reflect a weak, non-specific cis effect of the randomsequence used. Despite the possibility that insertion of sequences intothe 3′ UTR of an mRNA could exert a non-specific effect on mRNAfunction, it is nevertheless of interest, given that 293T cells expressa low level of endogenous anti-miR-30, but not miR-30, miRNA (FIG. 6A),that both indicator constructs predicted to be responsive to anti-miR30gave rise to significantly lower levels of luciferase than did thematched indicator plasmids specific for miR-30 (FIG. 6B, compare columns3 and 5 with 9 and 11). This observation is consistent with thehypothesis that these indicator constructs are subject to partialinhibition by the endogenous anti-miR-30 miRNA.

To gain insight into the mechanism of inhibition of luciferaseexpression documented in FIG. 6B, a northern analysis was next performedthat measured the level of expression of both the luc mRNA and the β-galinternal control mRNA (FIG. 6C). Consistent with the protein data, lucmRNA levels encoded by the indicator construct bearing random targetsites were unaffected by miR-30 or miR-21 expression, although they weresharply reduced by co-transfection of a previously described plasmid(Zeng et al, RNA 9:112-123 (2003)), termed pCMV-miR30-luc, that encodesan siRNA that is specific for the luc open reading frame (FIG. 6C, lanes2-4). An important observation emerged upon comparison of the luc mRNAexpression pattern in cultures transfected with indicator plasmidsbearing perfect versus bulged RNA targets. Specifically, while allcultures gave rise to detectable levels of the full-length, ˜2.3 kb lucmRNA, the cultures transfected with pCMV-miR-30 and indicator plasmidsbearing perfect targets were distinct in also giving rise to a secondluc mRNA band of ˜1.8 kb in size (FIG. 6C, lanes 8, 9 and 13). This isthe predicted size of the 5′ fragment of the full-length luc miRNA thatwould arise upon cleavage within the 3′UTR target sites (FIG. 5B) andtherefore suggests that both miR-30 (FIG. 6C, lanes 8 and 9) andanti-miR-30 (FIG. 6C, lane 13) are able to induce the specific cleavageof an mRNA bearing perfect target sites when overexpressed. Importantly,the lack of detectable cleavage of closely similar luc mRNAs bearingbulged target sites (FIG. 6C, lanes 6 and 11) is not due simply to alower level of inhibition, as the shorter luc mRNA band remained readilydetectable when RNA was prepared from cells co-transfected with theindicator construct bearing the perfect target sites together with a lowlevel of pCMV-miR-30 designed to mimic the level of inhibition seen whenthe target sites were bulged (compare lanes 6 and 8, FIG. 6C).

Cleavage of an mRNA by an Endogenous Human miRNA.

Unlike miR-30, but like the majority of miRNAs, processing of the miR-21pre-miRNA gives rise to only one stable mature miRNA (Lagos-Quintana,Science 294:853-858 (2001), Zeng et al, RNA 9:112-123 (2003)). AlthoughmiR-21 is expressed at readily detectable levels in 293T cells, thismiRNA (but not its putative antisense partner) can be overexpressed bytransfection of 293T cells with the pCMV-miR-21 expression plasmid (FIG.6A, lanes 5 and 6).

Indicator constructs analogous to pCMV-luc-Target-CAT, but containingeight copies of a perfect or bulged target specific for miR-21 (FIG.5A), were constructed and their biological activity analyzed. As shownin FIG. 7A, these constructs behaved similarly to the equivalentconstructs analyzed in FIG. 6A, in that both the bulged and perfecttarget sites supported specific inhibition by the co-transfectedpCMV-miR-21 effector plasmid, with the perfect indicator again beingsomewhat more responsive. Of note, the pCMV-luc-miR-21(P)-CAT indicatorconstruct gave rise to a quite low level of luc enzyme expression evenin the absence of a co-transfected effector plasmid, thus againsuggesting inhibition by endogenous miR-21 (FIG. 7A, lane 7).

Analysis of mRNA expression by northern blot analysis revealed readilydetectable levels of the ˜1.8 kb luc mRNA cleavage product in culturestransfected with the indicator construct bearing the miR-21(P) targetbut not the miR-21(B) target (FIG. 7B, lanes 2, 4 and 5), as previouslyalso seen with miR-30 (FIG. 6C). Importantly, however, this cleavageproduct was also readily detectable, albeit at a lower level, inpCMV-luc-miR-21(P)-CAT transfected cultures that were not co-transfectedwith pCMV-miR-21 (FIG. 6B, lane 6). The simplest explanation for thisobservation is that the endogenous miR-21 miRNA is responsible forcleavage of the miR-21(P) luc indicator mRNA within the fully homologoustarget sequence. In contrast, neither endogenous nor overexpressedmiR-21 is able to induce mRNA cleavage when this target bears a centralmismatch (FIG. 6C, lanes 2 and 3). Similarly, the low level ofendogenous anti-miR-30 miRNA (FIG. 6A) also gave rise to a low level ofcleavage of the mRNA encoded by the pCMV-luc-miR-30(AP)-CAT indicatorconstruct in some experiments, although the resultant mRNA cleavageproduct was present at levels only barely above background (FIG. 6C,lane 12).

Inhibition of mRNA Translation by a Synthetic siRNA.

Having established that both overexpressed and endogenous miRNAs cancleave target mRNAs, the next question was whether synthetic siRNAs caninhibit mRNA function without inducing mRNA cleavage. To address thisissue, two synthetic siRNAs specific for mRNAs encoding the DrosophilaNxt and Tap proteins were utilized. While these reagents can inhibitdNxt and dTap protein and mRNA expression in transfected Drosophila S2cells (Wiegand et al, Mol. Cell. Biol. 22:245-256 (2002)), these targetnucleotide sequences are not conserved in the human Nxt and Tap genes.

Indicator constructs based on pCMV-luc-Target, bearing perfect or bulgedtarget sequences homologous to the dNxt siRNA (FIG. 5A) were transfectedinto 293T cells along with either the dNxt or dTap siRNA (the latter asa negative control) and a β-gal expression plasmid. As shown in FIG. 8A,both the bulged and perfect dNxt target supported specific inhibition ofluc protein expression upon dNxt siRNA co-transfection, although theperfect target was again more responsive than the bulged target.Analysis of luc mRNA expression by northern blot revealed a drop in thelevel of full-length luc mRNA and the appearance of the predictedtruncated luc mRNA fragment in cultures transfected with the constructbearing the perfect dNxt target, even when inhibition of luc enzymeactivity was only a relatively modest ˜5 fold (FIG. 8B, lanes 7 and 8).In contrast, an equivalent ˜5 fold inhibition of the construct bearingthe bulged dNxt target failed to give rise to any detectable truncatedluc mRNA and indeed failed to significantly affect the level ofexpression the full length luc mRNA (FIG. 8B, lane 5). It was,therefore, concluded that the inhibition of luc enzyme expression seenwith the indicator construct bearing the bulged dNxt targets is due notto cleavage and degradation of the target luc mRNA but rather to someform of translational inhibition.

In summary, using entirely in vivo assays in human cells, it has beendemonstrated that endogenous human miR-21 miRNA, or overexpressed formsof the human miR-30 and anti-miR-30 miRNAs, can induce the cleavage ofmRNAs bearing fully complementary target sites, a phenotype previouslyviewed as characteristic of siRNAs (FIGS. 5 and 7). Conversely, it hasalso been demonstrated that a synthetic siRNA is able to downregulatethe expression of an mRNA bearing partially mismatched, bulged targetsites, without inducing detectable mRNA cleavage or reducing mRNAexpression levels (FIG. 8), an attribute previously viewed ascharacteristic of miRNAs (Hutv<gner et al, Curr. Opin. Genet. Dev.12:225-232 (2002))). Together, these data indicate that miRNAs andsiRNAs interact identically with mRNA molecules bearing target sites ofequivalent complementarity, i.e., in both cases perfect homology leadsto mRNA cleavage while a central bulge induces translational inhibition.These observations confirm and extend recent in vitro data documentingthe specific cleavage of an artificial RNA target by a cytoplasmicextract containing the human miRNA let-7 (Hutv<gner et al, Science297:2056-2060 (2002)).

Interpretation of the foregoing data was greatly facilitated by thefinding that the ˜2.3 kb luc mRNA encoded by the indicator constructsused gives rise to a stable ˜1.8 kb 5′ breakdown product after siRNA- ormiRNA-mediated cleavage at the introduced target sites. This RNAintermediate was invariably detected when a miRNA or siRNA encountered afully complementary artificial target but was never seen when the targetwas designed with a central mismatch (FIG. 6C, FIG. 7B and FIG. 8B).This RNA also differed from full-length luc mRNA in that only the latterwas detectable by Northern analysis when a probe specific for sequences3′ to the introduced target sites was tested. While the stability ofthis mRNA fragment is clearly fortuitous, others have previouslydetected the appearance of a stable luc mRNA cleavage intermediate incells treated with a luc-specific siRNA (Gitlin et al, Nature418:320-434 (2002)).

Although the data presented above demonstrate that miRNAs and siRNAs caninhibit mRNA expression by apparently identical mechanisms, it could beargued that siRNAs might still be more effective at RNA degradation thanat translation inhibition, while miRNAs might display the converseactivity. However, both for miRNAs and siRNAs, significantly moreeffective inhibition of luc enzyme activity was observed if the luc mRNAbore a fully complementary target and was therefore subject to RNAcleavage (FIGS. 6B, 7A and 8A). This could, of course, simply reflectmore efficient recruitment of miRNA- or siRNA-containingribonucleoprotein complexes to higher affinity RNA binding sites.However, while RISC appears to function as a true RNA cleavage enzymewhen presented with fully complementary RNA target sites (Hutvágner etal, Science 297:2056-2060 (2002)), it is speculated that target sitemismatches that preclude cleavage, such as a central RNA bulge, mayfreeze RISC in place on the mismatched RNA target. In this manner,centrally mismatched RNA targets may reduce the effective concentrationof their cognate RISC complex and thereby reduce the efficiency withwhich mRNA expression is inhibited.

All documents cited above are hereby incorporated in their entirety byreference.

What is claimed is:
 1. A method of inhibiting expression of a genecomprising introducing into a plant cell a DNA construct comprising aconstitutive or inducible promoter functional in said cell operablylinked to a nucleic acid comprising a nucleotide sequence encoding, aspart of a longer encoded sequence, an miRNA precursor, said miRNAprecursor comprising a stem loop structure and comprising in said stemof said stem loop structure a sequence complementary to a portion of anRNA transcript of said gene, wherein said stem of said stem loopstructure is about 19-45 base pairs long and the loop of said stem loopstructure is about 4-25 nucleotides, wherein, following introduction ofsaid construct into said cell: (i) said nucleotide sequence istranscribed, (ii) the resulting transcript of said nucleotide sequenceis processed so that said miRNA precursor is excised from saidtranscript of said nucleotide sequence, (iii) said miRNA precursor isprocessed so that a mature miRNA about 21 or 22 nucleotides in length isexcised from said miRNA precursor, and (iv) inhibition of expression ofsaid gene is effected.
 2. The method according to claim 1, wherein saidloop of said stem loop structure of said miRNA precursor comprises asequence corresponding to a naturally occurring miRNA.
 3. The methodaccording to claim 1, wherein the base of said stem of said stem loopstructure of said miRNA precursor comprises a basepaired region at least3 base pairs in length.
 4. The method according to claim 1, wherein saidpromoter is a polymerase II-based promoter.
 5. The method according toclaim 1, wherein said cell is a cultured cell.
 6. The method accordingto claim 1, wherein said mature miRNA induces degradation of said RNAtranscript of said gene.
 7. The method according to claim 6, whereinsaid RNA transcript of said gene is an mRNA.
 8. The method according toclaim 1, wherein said stem of said stem loop structure of said miRNAprecursor includes at least one bulge.
 9. The method according to claim1, wherein said loop of said stem loop structure of said miRNA precursorcomprises 6-15 nucleotides.